RESUMO
The fusion of autophagosomes and lysosomes is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). Here, we generate a hepatocyte-specific CHIP knockout (H-KO) mouse model that develops NAFLD more rapidly in response to a high-fat diet (HFD) or high-fat, high-fructose diet (HFHFD). The accumulation of P62 and LC3 in the livers of H-KO mice and CHIP-depleted cells indicates the inhibition of autophagosome-lysosome fusion. AAV8-mediated overexpression of CHIP in the murine liver slows the progression of NAFLD induced by HFD or HFHFD feeding. Mechanistically, CHIP induced K63- and K27-linked polyubiquitination at the lysine 198 residue of STX17, resulting in increased STX17-SNAP29-VAMP8 complex formation. The STX17 K198R mutant was not ubiquitinated by CHIP; it interfered with its interaction with VAMP8, rendering STX17 incapable of inhibiting steatosis development in mice. These results indicate that a signaling regulatory mechanism involving CHIP-mediated non-degradative ubiquitination of STX17 is necessary for autophagosome-lysosome fusion.
Assuntos
Autofagossomos , Lisossomos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Lisossomos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Autofagossomos/metabolismo , Camundongos , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Hepatócitos/metabolismo , Modelos Animais de Doenças , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusão de Membrana , Autofagia , Fator de Transcrição TFIIHRESUMO
The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6 kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6 kb intronic sequence.
Assuntos
Alelos , Variações do Número de Cópias de DNA , Cor de Cabelo , Íntrons , Melanoma , Proteínas Qa-SNARE , Cavalos/genética , Animais , Variações do Número de Cópias de DNA/genética , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Melanoma/genética , Melanoma/veterinária , Melanoma/epidemiologia , Íntrons/genética , Cor de Cabelo/genética , Linhagem , Masculino , Feminino , Fenótipo , Incidência , Doenças dos Cavalos/genética , Doenças dos Cavalos/epidemiologia , Pigmentação da Pele/genéticaRESUMO
Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.
Assuntos
Proteínas Qa-SNARE , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Animais , Camundongos , Humanos , Proteínas Priônicas/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/química , Neurônios/metabolismo , Agregados Proteicos , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/genéticaRESUMO
Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.
Assuntos
Neurônios , Proteínas Qa-SNARE , Proteína 25 Associada a Sinaptossoma , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/química , Neurônios/metabolismo , Animais , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/química , Cinética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Ratos , Multimerização ProteicaRESUMO
OBJECTIVE: Neonatal sepsis and familial hemophagocytic lymphohistiocytosis (fHLH) have similar clinical and laboratory symptoms and the possibility of overlooking fHLH diagnosis is high in newborns with sepsis. History of consanguineous marriage and/or sibling death, hepatomegaly/splenomegaly, and hyperferritinemia (>500 ng/mL) are likely to support fHLH in newborns with sepsis. Therefore, in newborns with sepsis in whom at least 2 of these 3 criteria were detected, genetic variants was investigated for the definitive diagnosed of fHLH. According to the results of genetic examination, we investigated whether these criteria supporting fHLH could be used as a screening test in fHLH. MATERIALS AND METHODS: fHLH-associated genetic variants were investigated in 22 patients diagnosed with neonatal sepsis who fulfilled at least 2 of the following criteria (1) history of consanguineous marriage and/or sibling death, (2) hepatomegaly/splenomegaly, and (3) hyperferritinemia (>500 ng/mL). RESULTS: Heterozygous variants were determined in 6 patients (27.2%): 3 STXBP2 , 1 STX11 , 1 UNC13D , and 1 PRF1 . Polymorphisms associated with the clinical symptoms and signs of HLH were determined in 5 patients (22.7%): 4 UNC13D , 1 PRF1 . Two patients were in the heterozygous variants and polymorphism associated with the clinical symptoms and signs of HLH groups. In 12 patients, benign polymorphisms were detected in STXBP2 and UNC13D genes. No change in fHLH associated genes were found in 1 patient. CONCLUSION: Some variants and/or polymorphisms identified in our patients have been previously reported in patients with HLH. Therefore, we recommend further investigation of fHLH in patients with neonatal sepsis who fulfill at least 2 of the above 3 criteria.
Assuntos
Linfo-Histiocitose Hemofagocítica , Sepse Neonatal , Humanos , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/diagnóstico , Recém-Nascido , Masculino , Feminino , Sepse Neonatal/diagnóstico , Sepse Neonatal/genética , Perforina/genética , Proteínas Qa-SNARE/genética , Proteínas de Membrana/genética , Testes Genéticos/métodosRESUMO
Syntaxin 17 (STX17) has been identified as a crucial factor in mediating the fusion of autophagosomes and lysosomes. However, its specific involvement in the context of atherosclerosis (AS) remains unclear. This study sought to elucidate the role and mechanistic contributions of STX17 in the initiation and progression of AS. Utilizing both in vivo and in vitro AS model systems, we employed ApoE knockout (KO) mice subjected to a high-fat diet and human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) to assess STX17 expression. To investigate underlying mechanisms, we employed shRNA-STX17 lentivirus to knock down STX17 expression, followed by evaluating autophagy and inflammation in HUVECs. In both in vivo and in vitro AS models, STX17 expression was significantly upregulated. Knockdown of STX17 exacerbated HUVEC damage, both with and without ox-LDL treatment. Additionally, we observed that STX17 knockdown impaired autophagosome degradation, impeded autophagy flux and also resulted in the accumulation of dysfunctional lysosomes in HUVECs. Moreover, STX17 knockdown intensified the inflammatory response following ox-LDL treatment in HUVECs. Further mechanistic exploration revealed an association between STX17 and STING; reducing STX17 expression increased STING levels. Further knockdown of STING enhanced autophagy flux. In summary, our findings suggest that STX17 knockdown worsens AS by impeding autophagy flux and amplifying the inflammatory response. Additionally, the interaction between STX17 and STING may play a crucial role in STX17-mediated autophagy.
Assuntos
Aterosclerose , Autofagia , Células Endoteliais da Veia Umbilical Humana , Inflamação , Lipoproteínas LDL , Proteínas Qa-SNARE , Autofagia/genética , Animais , Humanos , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Camundongos , Lipoproteínas LDL/metabolismo , Técnicas de Silenciamento de Genes , Lisossomos/metabolismo , Camundongos Knockout , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Dieta Hiperlipídica/efeitos adversos , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/deficiênciaRESUMO
OBJECTIVE: Our objective was to evaluate whether there is an enrichment of rare variants in familial hemophagocytic lymphohistiocytosis (HLH)-associated genes among patients with systemic juvenile idiopathic arthritis (sJIA) with or without macrophage activation syndrome (MAS). METHODS: Targeted sequencing of HLH genes (LYST, PRF1, RAB27A, STX11, STXBP2, UNC13D) was performed in patients with sJIA from an established cohort. Sequence data from control participants were obtained in silico (database of Genotypes and Phenotypes: phs000280.v8.p2). Rare variant association testing (RVT) was performed with sequence kernel association test package. Significance was defined as P < 0.05 after 100,000 permutations. RESULTS: Sequencing data from 524 sJIA cases were jointly called and harmonized with exome-derived target data from 3,000 controls. Quality control operations produced a set of 480 cases and 2,924 ancestrally matched control participants. RVT of cases and controls revealed a significant association with rare protein-altering variants (minor allele frequency [MAF] < 0.01) of STXBP2 (P = 0.020) and ultrarare variants (MAF < 0.001) of STXBP2 (P = 0.006) and UNC13D (P = 0.046). A subanalysis of 32 cases with known MAS and 90 without revealed a significant difference in the distribution of rare UNC13D variants (P = 0.0047) between the groups. Additionally, patients with sJIA more often carried two or more HLH variants than did controls (P = 0.007), driven largely by digenic combinations involving LYST. CONCLUSION: We identified an enrichment of rare HLH variants in patients with sJIA compared with controls, driven by STXBP2 and UNC13D. Biallelic variation in HLH genes was associated with sJIA, driven by LYST. Only UNC13D displayed enrichment in patients with MAS. This suggests that HLH variants may contribute to the pathophysiology of sJIA, even without MAS.
Assuntos
Artrite Juvenil , Linfo-Histiocitose Hemofagocítica , Síndrome de Ativação Macrofágica , Proteínas de Membrana , Proteínas Munc18 , Perforina , Proteínas Qa-SNARE , Humanos , Linfo-Histiocitose Hemofagocítica/genética , Artrite Juvenil/genética , Proteínas Qa-SNARE/genética , Proteínas de Membrana/genética , Proteínas Munc18/genética , Perforina/genética , Masculino , Feminino , Criança , Síndrome de Ativação Macrofágica/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas de Membrana Lisossomal/genética , Proteínas R-SNARE/genética , Pré-Escolar , Estudos de Casos e Controles , Proteínas rab de Ligação ao GTP/genética , Predisposição Genética para Doença , Adolescente , Variação Genética , Proteínas de Transporte VesicularRESUMO
During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
Assuntos
Autofagossomos , Fosfatos de Fosfatidilinositol , Proteínas Qa-SNARE , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Autofagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Humanos , Simulação de Dinâmica Molecular , Autofagia/fisiologiaRESUMO
Syntaxin5 (Syx5) belongs to SNAREs family, which play important roles in fusion of vesicles to target membranes. Most of what we know about functions of Syx5 originates from studies in fungal or vertebrate cells, how Syx5 operates during the development of insects is poorly understood. In this study, we investigated the role of LmSyx5 in the gut development of the hemimetabolous insect Locusta migratoria. LmSyx5 was expressed in many tissues, with higher levels in the gut. Knockdown of LmSyx5 by RNA interference (RNAi) considerably suppressed feeding in both nymphs and adults. The dsLmSyx5-injected locusts lost body weight and finally died at a mortality of 100%. Furthermore, hematoxylin-eosin staining indicated that the midgut is deformed in dsLmSyx5-treated nymphs and the brush border in midgut epithelial cells is severely damaged, suggesting that LmSyx5 is involved in morphogenesis of the midgut. TEM further showed that the endoplasmic reticulum of midgut cells have a bloated appearance. Taken together, these results suggest that LmSyx5 is essential for midgut epithelial homeostsis that affects growth and development of L. migratoria. Thus, Syx5 is a promising RNAi target for controlling L. migratoria, and even other pests.
Assuntos
Comportamento Alimentar , Proteínas de Insetos , Mucosa Intestinal , Locusta migratoria , Proteínas Qa-SNARE , Locusta migratoria/genética , Locusta migratoria/crescimento & desenvolvimento , Locusta migratoria/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Mucosa Intestinal/crescimento & desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Comportamento Alimentar/fisiologia , Técnicas de Silenciamento de Genes , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Peso Corporal/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
By modulating stomatal opening and closure, plants control gas exchange, water loss, and photosynthesis in response to various environmental signals. During light-induced stomatal opening, the transport of ions and solutes across the plasma membrane (PM) of the surrounding guard cells results in an increase in turgor pressure, leading to cell swelling. Simultaneously, vesicles for exocytosis are delivered via membrane trafficking to compensate for the enlarged cell surface area and maintain an appropriate ion-channel density in the PM. In eukaryotic cells, soluble N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAREs) mediate membrane fusion between vesicles and target compartments by pairing the cognate glutamine (Q)- and arginine (R)-SNAREs to form a core SNARE complex. Syntaxin of plants 121 (SYP121) is a known Q-SNARE involved in stomatal movement, which not only facilitates the recycling of K+ channels to the PM but also binds to the channels to regulate their activity. In this study, we found that the expression of a receptor-like cytoplasmic kinase, low-K+ sensitive 4/schengen 1 (LKS4/SGN1), was induced by light; it directly interacted with SYP121 and phosphorylated T270 within the SNARE motif. Further investigation revealed that LKS4-dependent phosphorylation of SYP121 facilitated the interaction between SYP121 and R-SNARE vesicle-associated membrane protein 722 (VAMP722), promoting the assembly of the SNARE complex. Our findings demonstrate that the phosphorylation of SNARE proteins is an important strategy adopted by plants to regulate the SNARE complex assembly as well as membrane fusion. Additionally, we discovered the function of LKS4/SGN1 in light-induced stomatal opening via the phosphorylation of SYP121.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Luz , Estômatos de Plantas , Proteínas Qa-SNARE , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Estômatos de Plantas/fisiologia , Estômatos de Plantas/metabolismo , Estômatos de Plantas/efeitos da radiação , Fosforilação , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Proteínas de Ciclo CelularRESUMO
SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.
Assuntos
Linfócitos B , Linfócitos T CD4-Positivos , Proteínas Qa-SNARE , Animais , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Camundongos , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Feminino , Masculino , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunidade Humoral , ExocitoseRESUMO
Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.
Assuntos
Fusão de Membrana , Secretagoginas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Secretagoginas/metabolismo , Membrana Celular/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Exocitose , Comunicação Celular , Sintaxina 1/metabolismo , Ligação ProteicaRESUMO
The Grey allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the Syntaxin 17 gene. A recent study demonstrated that the most common allele at the Grey locus (G3) involves three tandem copies of this sequence, whilst a more rare allele (G2) has two tandem copies and the wild-type allele (G1) only one copy. The G3 allele is causing fast greying and high incidence of skin melanoma, whereas the G2 allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented in melanoma tissue from Grey horses. Functional studies showed that this intronic sequence acts as a weak melanocyte-specific enhancer that becomes substantially stronger by the copy number expansion. The Grey mutation is associated with upregulated expression of both Syntaxin 17 and the neighbouring NR4A3 gene in Grey horse melanomas. It is still an open question which of these genes is most important for the phenotypic effects or if causality is due to the combined effect of upregulation of both genes. Interestingly, RNAseq data in the Human Protein Atlas give support for a possible role of NR4A3 because it is particularly upregulated in human skin cancer, and it belongs to a cluster of genes associated with skin cancer and melanin biosynthesis. The Grey mutation and its association with melanoma provide a possibility to study the path to tumour development in numerous Grey horses carrying exactly the same predisposing mutation.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/genética , Melanoma/veterinária , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/veterinária , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Mutação , Cabelo/metabolismo , Cabelo/patologiaRESUMO
Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.
Assuntos
Proteína Semelhante a ELAV 1 , Macrófagos , MicroRNAs , Proteínas Qa-SNARE , Animais , Humanos , Camundongos , Endossomos/metabolismo , Leishmania donovani/metabolismo , Leishmania donovani/genética , Ativação de Macrófagos , Macrófagos/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Transporte de RNA , Proteína Semelhante a ELAV 1/metabolismoRESUMO
As a primary liver malignancy, hepatocellular carcinoma (HCC) is commonly induced by chronic liver disease and cirrhosis. Bioinformatics analysis reveals that long noncoding RNA KDM4A antisense RNA 1 (KDM4A-AS1) may be aberrantly expressed in HCC and its abnormal expression might influence prognosis in patients. We conducted this study to illustrate the functions and mechanism of KDM4A-AS1 in regulating HCC malignant cell behavior. KD-M4A-AS1, microRNA (miR)-4306 and messenger RNA syntaxin 6 (STX6) expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). HCC cell proliferation, apoptosis, migration, and invasion were measured by colony forming assays, flow cytometry, wound healing and Transwell assays. The interaction between genes was verified by RNA immunoprecipitation and luciferase reporter assays. Western blotting was performed to quantify protein expression of STX6 or apoptotic markers. KDM4A-AS1 was highly expressed in HCC cells and tissues. KDM4A-AS1 knockdown led to enhanced HCC cell apoptosis and suppressed HCC cell proliferation, migration, and invasion. MiR-4306 bound to and negatively regulated STX6. KDM4A-AS1 directly bound to miR-4306 and thus up-regulated STX6. STX6 overexpression reversed the inhibitory influence of KDM4A-AS1 depletion on HCC malignant behavior. KDM4A-AS1 promotes HCC cell migration, invasion, and growth by upregulating STX6 via miR-4306.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , RNA Longo não Codificante , Humanos , Carcinoma Hepatocelular/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismoRESUMO
Acinetobacter baumannii (A. baumannii) causes autophagy flux disorder by degrading STX17, resulting in a serious inflammatory response. It remains unclear whether STX17 can alter the inflammatory response process by controlling autolysosome function. This study aimed to explore the role of STX17 in the regulation of pyroptosis induced by A. baumannii. Our findings indicate that overexpression of STX17 enhances autophagosome degradation, increases LAMP1 expression, reduces Cathepsin B release, and improves lysosomal function. Conversely, knockdown of STX17 suppresses autophagosome degradation, reduces LAMP1 expression, augments Cathepsin B release, and accelerates lysosomal dysfunction. In instances of A. baumannii infection, overexpression of STX17 was found to improve lysosomal function and reduce the expression of mature of GSDMD and IL-1ß, along with the release of LDH, thus inhibiting pyroptosis caused by A. baumannii. Conversely, knockdown of STX17 led to increased lysosomal dysfunction and further enhanced the expression of mature of GSDMD and IL-1ß, and increased the release of LDH, exacerbating pyroptosis induced by A. baumannii. These findings suggest that STX17 regulates pyroptosis induced by A. baumannii by modulating lysosomal function.
Assuntos
Acinetobacter baumannii , Interleucina-1beta , Lisossomos , Piroptose , Proteínas Qa-SNARE , Lisossomos/metabolismo , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Humanos , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Autofagia , Animais , Catepsina B/metabolismo , Catepsina B/genética , Infecções por Acinetobacter/microbiologia , Camundongos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Autofagossomos/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , GasderminasRESUMO
In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Qa-SNARE , Estresse Salino , Aquaporinas/metabolismo , Aquaporinas/genética , Arabidopsis/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Transporte Proteico , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genéticaRESUMO
HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-α (TNFα) secretion and that Gag and TNFα are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNFα secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNFα and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNFα directly binds the C-terminal domain of Syx6. Altogether, our data provide evidence that both Gag and TNFα make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNFα secretion in HIV-1 infection.
Assuntos
HIV-1 , Proteínas Qa-SNARE , Vesículas Transportadoras , Fator de Necrose Tumoral alfa , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Endossomos/metabolismo , HIV-1/genética , HIV-1/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transporte Proteico/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Ligação Proteica , Domínios Proteicos , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Linhagem Celular , Vesículas Transportadoras/metabolismo , Replicação Viral/genéticaRESUMO
Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is thought to occur when the cellular prion protein (PrPC) spontaneously misfolds and assembles into prion fibrils, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD, we recently identified risk variants in and around the gene STX6, with evidence to suggest a causal increase of STX6 expression in disease-relevant brain regions. STX6 encodes syntaxin-6, a SNARE protein primarily involved in early endosome to trans-Golgi network retrograde transport. Here we developed and characterised a mouse model with genetic depletion of Stx6 and investigated a causal role of Stx6 expression in mouse prion disease through a classical prion transmission study, assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation periods and prion-related neuropathology. Following inoculation with RML prions, incubation periods in Stx6-/- and Stx6+/- mice differed by 12 days relative to wildtype. Similarly, in Stx6-/- mice, disease incubation periods following inoculation with ME7 prions also differed by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculated Stx6-/- animals and a variable effect of Stx6 expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly, Stx6-/- mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide some support for a pathological role of Stx6 expression in prion disease, which warrants further investigation in the context of prion disease but also other neurodegenerative diseases considering syntaxin-6 appears to have pleiotropic risk effects in progressive supranuclear palsy and Alzheimer's disease.
Assuntos
Síndrome de Creutzfeldt-Jakob , Doenças Priônicas , Príons , Camundongos , Humanos , Animais , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patologia , Príons/genética , Príons/metabolismo , Estudo de Associação Genômica Ampla , Camundongos Transgênicos , Encéfalo/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismoRESUMO
BACKGROUND: Some conventional prognostic biomarkers for esophageal squamous cell carcinoma (ESCC) have the disadvantage that they have only been investigated at the level of either mRNA or protein levels or only in individual cohorts. Associations between Syntaxin 3 (STX3) expression and malignancy have been reported in several tumor types but not in ESCC. Here, we investigated the levels of both STX3 mRNA and protein, and its prognostic potential in two independent cohorts of patients with ESCC. METHODS: STX3 mRNA levels were examined in surgical specimens by quantitative PCR in a cohort that included 176 ESCC patients. STX3 protein levels were investigated in surgically resected ESCC tissues by immunohistochemistry using tissue microarrays in a different cohort of 177 ESCC patients. Correlations were analyzed between the expression of STX3 mRNA and protein with clinicopathological factors and long-term prognosis. RESULTS: Quantitative PCR indicated a significant association between high level of STX3 mRNA expression and lymph node involvement, pathological stage, and poor overall survival. The multivariate analysis demonstrated that high STX3 mRNA expression was independently associated with poor overall survival outcomes. Immunohistochemistry revealed that STX3 protein expression in ESCC tissues and high STX3 protein expression were also significantly correlated with unfavorable overall survival. CONCLUSIONS: Overexpression of STX3 mRNA and protein may serve as potential prognostic biomarkers for ESCC patients.